松材线虫RAPD规范化反应体系的构建

 针对随机扩增DNA多态性(RAPD)低重复性的特点,在研究松材线虫遗传多样性的过程中,对可能影响RAPD扩增结果的PCR仪型号,模板DNA,Mg²⁺,TaqDNA聚合酶引物及dNTP浓度等因素进行了实验探索,以获得最佳反应条件,构建稳定的松材线虫RAPD扩增反应体系,结果发现上述影响因子在相当大的范围内对RAPD扩增结果的影响有限,进而对在不同实验室间的RAPD结果缺乏重复性和可比性的原因进行了讨论,认为模板DNA的污染是造成这一现象的重要原因,并在此基础上提出了相应的规范化操作建议.

Establishment of Optimal Conditions for Bursaphelenchus xylophilus RAPD Analysis

The low reproducibility is the great disadvantage of RAPD (random amplified polymorphic DNA) method. In view of this situation, some essential factors (such as the type of PCR amplifier, concentration of Mg 2 , dNTP, Taq DNA polymerase, primers and template DNA), which were considered to affect the result of RAPD assay, were probed in the process of the analysis of the genetic diversity among the populations of Bursaphelenchus xylophilus . As the result, a reliable and steady procedure has been developed for Bursaphelenchus xylophilus RAPD assay. It also can be found that those factors had little influence on amplification in a wide range. Furthermore, the reasons of poor reprducibility and comparition in RAPD application were discussed. Then some practical methods of operation in Bursaphelenchus xylophilus RAPD analysis were commanded.